ANTHELMINTIC EFFICACY OF PELLETED CRANBERRY VINE AGAINST GASTROINTESTINAL NEMATODES OF SHEEP

Gastrointestinal nematodes (GIN) represent a major health concern in small ruminants as well as a constraint for producers. The prevalence of anthelmintic resistance in small ruminant GIN has increased rapidly worldwide, creating the need for alternative control methods to address the issue. The discovery that plant secondary compounds, including proanthocyanidins (PAC), suppress GIN infections has provided promise for alternative methods of GIN control. Although the mechanism is not well understood, studies have shown in vitro and in vivo evidence of anti-parasitic effects of bioactive plants. Recently a study from our laboratory provided in vitro evidence of anthelmintic activity of cranberry vine (CV) PAC organic extracts as well as CV aqueous extracts (CV-AqE) against Haemonchus contortus first stage larvae (L1) and adult worm motility and some activity of CV-AqE against egg hatching. This previous study also showed a slight suppression of fecal egg count (FEC) in vivo when chopped cranberry was fed to parasitized lambs. The current study was designed to further investigate the effects of CV on GIN infections in sheep. This study utilized two feeding trials in order to investigate the in vivo effects of CV. In the first feeding trial, lambs experimentally infected with Haemonchus contortus were given a 100% CV pellet (CVP) as a supplement to their regular grain. Although the palatability and consumption of the pellet was a major issue, supplementing the CVP into the diet appeared to suppress the FEC compared to untreated sheep at the end of the six-week trial, with no difference observed in packed cell volume or total worm burden. In the second feeding trial, lambs with a mixed natural and experimental GIN infection were fed two levels of a 50% cranberry vine pellet that had been formulated to contain equivalent amounts of digestible dry matter. Under the conditions of the study, the FEC of the lambs consuming 500g of CV per day did not change over the course of the study unlike the control group and the lambs consuming 250g of CV per day. Although further research is needed to better understand this phenomenon, it is suggested that CV may have potential as a viable and sustainable option for northeast sheep producers to utilize as a strategy for GIN control.

x    Historically, sheep meat was a by-product of the wool industry; however, wool production has seen a steady decline due to the introduction of synthetic fibers, causing a surge in lamb production and consumption (Chikwanha et al., 2018). The decline in the wool industry has also caused an increase in hair sheep which require little to no shearing while still producing a high-quality carcass (Aldridge et al., 2018). Although sheep meat consumption and production lags behind beef, pork, and poultry, there has been an increase in consumption, a trend expected to continue due to population growth and growing incomes (Montossi et al., 2013;Chikwanha et al., 2018). In general, meat consumption patterns vary greatly due to socio-economic and cultural trends, as well as the lifestyles of diverse groups of consumers (Bernués et al., 2012). Sheep meat has an intense and distinct species flavor and aroma which also contributes to the variability of consumption, especially across different countries (Schönfeldt et al., 1993).

Economic impact
The In the US market, sheep and lamb production and related agricultural products amounted to over $1 billion in total economic contribution (USDA, National Agricultural Statistics Service, 2019).

Production losses
In the United States and globally, gastrointestinal nematode infection has been identified as the costliest disease to affect the sheep industry, representing great economic importance for producers Qamar et al., 2011). Sheep are able to graze in areas that are not well suited for most other species and in conjunction help to prevent wildfires by grazing brush and vegetation (USDA, 2011;Aldridge et al., 2018); however, gastrointestinal nematode (GIN) infections have been identified as one of the major limitations faced in pasture-raised production of small ruminants .
The costs associated with GIN are twofold, and are comprised of both direct and indirect expenses. Direct expenses are those associated with decreases in production, decreases in gain, and host mortality; the indirect expenses are those such as treatment costs and increased labor (Torres-Acosta and Hoste, 2008).

Gastrointestinal nematodes of small ruminants Introduction
Gastrointestinal helminth infections are the most prevalent parasitic diseases affecting small ruminants worldwide (Torres-Acosta and Hoste, 2008; Traversa and von Samson-Himmelstjerna, 2016). Haemonchus contortus (the barber pole worm), Teladorsagia circumcincta (the brown stomach worm) and Trichostrongylus colubriformis (the bankrupt worm) are the most abundant gastrointestinal nematodes in sheep, and account for the greatest amount of economic and production losses (O'Connor et al., 2006). The infections can have both direct (mortality, decreased production) and indirect (treatment costs, labor, housing) impacts (Torres-Acosta and Hoste, 2008), making them a major constraint for sheep producers. The life cycle of these three parasites is similar, although they each vary in developmental requirements, prepatent periods, and predilection sites.

Life cycle
Trichostrongyle nematodes (of the suborder Trichostrongylina) are parasites affecting the gastrointestinal tract and lungs of pasture-raised animals, as well as wild animals and birds . Pastures become infected with GIN when eggs are shed in the feces of grazing animals.
Fertilized female worms found in the gastrointestinal tract can lay 200-10,000 eggs per day, depending on the species , contributing to rapid pasture contamination. The larvae hatch from the eggs within the feces as the L1 stage and then develop into L2 and L3 larvae within the feces. The infective L3 larvae migrate from the feces onto the pasture where they can be readily consumed by grazing animals (Figure 1).
The L3 larvae is ensheathed within a cuticular outer layer which helps to protect them from the environment but also prevents them from feeding . Once inside the host, the larvae undergo a process known as exsheathment where the protective sheath is lost and they can mature into adult worms (Nikolaou and Gasser, 2006). The L4 and adult stages of the worm are parasitic inside the host. Mature female worms will begin to lay eggs approximately two to four weeks after infection, thus continuing the cycle through pasture contamination.   Nikolaou and Gasser 2006;Sommerville and Davey 2002).

Haemonchus contortus
Of the three most common GIN of small ruminants, Haemonchus contortus is the most pathogenic species causing issues such as anemia, reduced nutrient absorption, production losses, and in severe cases death (Veríssimo et al., 2012;. Haemonchus contortus is a blood-feeding parasite which inhabits the abomasum (the fourth chamber of the ruminant stomach), and one adult worm can consume up to 30 µL of blood each day . The abomasum is also the site where adult worms mate and produce eggs. Unique to H.
contortus is the high level of fecundity in females; one adult female alone can lay between 5000-15,000 eggs per day . The eggs are then shed in the feces of the animal leading to rapid pasture contamination even with low infection levels, thus continuing the parasite life cycle.
Young lambs and periparturient ewes are particularly susceptible to overwhelming worm burdens and anemia (Santos et al., 2014), although any sheep infected heavily can be subject to hemorrhage of the abomasum and potentially fatal anemia (Ortolani et al., 2013). Due to the pathogenicity of these parasites, proper and effective control methods are needed to ensure good health and adequate production levels for farmers.

Teladorsagia circumcincta and Trichostrongylus colubriformis
Sheep infected with Teladorsagia circumcincta are challenged with increased fluid secretion and increased flow rate of digesta at the abomasum, although most of this can be reabsorbed in the distal parts of the small intestine (Williams et al., 2010). Trichostrongylus colubriformis infection increases the flow of protein and minerals through the small intestine, blocking absorption and thereby increasing the animal's energy demand (Bown et al., 1991). Both T. circumcincta and T. colubriformis can cause immune-mediated diarrhea in infected animals, and a mixed infection can lead to increased inflammatory cells in the small intestine (Williams et al., 2010). Decreased nutrient absorption and diarrhea associated with these GI worms lead to decreased gain and production losses. As previously mentioned, the life cycle of T. circumcincta and T. colubriformis are similar to H. contortus, but diarrhea or scouring in infected animals is an observable clinical symptom which is generally not associated with H. contortus, unless in mixed infections .

Current control options
Commercially produced anthelmintics The use of broad-spectrum anthelmintics has been the primary strategy for treatment of GIN in small ruminants for many years. The anthelmintic drugs available for use in small ruminants belong to three broad classes: benzimidazoles (including albendazole and fenbendazole), cholinergic agonists (such as levamisole), and macrocyclic lactones (including ivermectin and moxidectin) . These drugs are inexpensive, safe, and are generally effective across a broad spectrum (Zajac et al., 2000;McKellar and Jackson, 2004;Ketzis et al., 2006) and many farmers choose to treat all of their animals prophylactically every 3-4 weeks regardless of infection level in order to promote productivity and increase profits (da Cruz et al., 2010;.

Anthelmintic resistance
The overuse and improper use of commercial anthelmintics have led to the development and increasing prevalence of multiple drug resistance in H.
contortus and other GIN worldwide, creating the need for alternative control methods .
Additionally, there is a push by the public to move away from traditional chemical anthelmintics due to the possibility that the synthetic drugs can leave residues in animal products as well as accumulate in the environment (Ketzis et al., 2006;Silveira et al., 2012). Parasites that are resistant to multiple or all available anthelmintic drugs are becoming increasingly more prevalent all over the world .
In the United States and globally, drug resistant and multiple drug resistant nematodes are now widespread Crook et al., 2016). Resistance has been reported to all classes of anthelmintic drugs in several parasite species, and some isolates have been identified as resistant to all drug classes; drugs which were once greater than 99.9% effective have decreased to 0-70% effective (Sangster, 2001). Anthelmintic resistance and multiple drug resistance has been reported across the United States Crook et al., 2016) as well as Canada (Falzon et al., 2013), Australia Leathwick andBesier, 2014), New Zealand (McKenna, 2010), South Africa (Tsotetsi et al., 2013), Scotland , Brazil (da Cruz et al., 2010;Veríssimo et al., 2012) and across multiple countries in Europe (Geurden et al., 2014;Traversa and von Samson-Himmelstjerna, 2016;Ploeger and Everts, 2018).
Genetic variability in parasites leads to anthelmintic resistance, and human actions such as the overuse of anthelmintics contributes to more rapid accumulation of this resistance (da Cruz et al., 2010). Gastrointestinal nematodes of small ruminants have a number of genetic characteristics contributing to their resistance; high fecundity rates result in rapid nucleotide sequence evolution and there are high levels of gene flow due to host movement from farm to farm, helping to not only select for resistant genes but also to spread the resistant populations (Sangster, 2001;.

Screening methods (in vitro/in vivo)
The worldwide emergence of anthelmintic resistant parasites has motivated the investigation into alternative control options for gastrointestinal nematodes. Preliminary screening is done through in vitro testing with controlled laboratory settings. In vitro assays have several advantages such as low cost and rapid turnover which allows for multiple compounds to be tested simultaneously (Githiori et al., 2006). Several in vitro assays are commonly used in laboratories in order to test the anthelmintic activity of potential alternative treatment candidates (Marie-Magdeleine et al., 2009). The in vitro assays commonly employed for this testing include the egg hatch assay (Hubert and Kerboeuf, 1984), larval development assay (Hubert and Kerboeuf, 1992), larval migration inhibition assay (Wagland et al., 1992;Rabel et al., 1994), adult worm motility assay (Hounzangbe-Adote et al., 2005), and exsheathment assay (Conder and Johnson 1996). The hypothesis is that nematicidal activity observed in vitro will correspond to activity and then be reproducible in vivo (Marie-Magdeleine et al., 2009).

Copper oxide wire particles
Copper oxide wire particles (COWP) are a product marketed as a livestock supplement for animals living in copper deficient areas . The COWP are given orally as a bolus containing a designated amount based on species requirements . The COWP become lodged in the abomasal mucosa of the animal allowing for extended copper release . Studies have been conducted to determine whether the copper supplementation from the copper oxide wire particles has an effect on the establishment of gastrointestinal nematode infections in sheep or goats. In a study conducted by (Bang et al., 1990) in 1990, a 96% reduction of H. contortus and a 56% reduction of T. circumcincta establishment was observed in lambs treated with 5g COWP compared to untreated control lambs, with no significant effect on T. colubriformis (Bang et al., 1990). Several studies have shown reduction in both fecal egg counts and adult worm burdens in H. contortus infections in sheep (Knox, 2002; as well as goats (Chartier et al., 2000 contortus as well as a 74% reduction in T. circumcincta when compared to untreated control lambs (Waller et al., 2004). Interestingly, it has also been shown that COWP combined with a traditional treatment of albendazole increases the efficacy against several GIN genera when compared to either treatment given separately , providing an effective combination treatment which could be useful in anthelmintic resistant parasite populations.
Although COWP have shown anthelmintic activity and reduced establishment of GIN in multiple studies, the potential use is limited. Copper must be used cautiously in sheep because they are at high risk for copper toxicosis when levels build up in the liver (Terrill et al., 2012;; for this reason only one minimal dose per year is recommended . Goats are browsers and have a higher tolerance for copper compared to sheep, as browse plants tend to have higher copper levels . For this reason, toxicosis is likely not a concern in goats, and they could potentially receive higher or more frequent doses than sheep could .

Nematophagous Fungi
Nematophagous fungi, such as Duddingtonia flagrans, have also been extensively studied as a form of biological control to target the free-living stages of parasites. Duddingtonia flagrans is a predacious fungus which has the ability to trap and destroy nematode larvae (Larsen et al., 1998;. Most research related to biological control of GIN through the use of fungi has focused on D. flagrans due to its particular ability to produce abundant, thick-walled intercalary chlamydospores which allow for passage through the gut of grazing livestock (Waller et al., 1994;Larsen et al., 1998). When spores are fed to animals they are deposited in the feces of the animal after passing through the digestive tract, where they can then trap and kill larvae in order to reduce pasture contamination Terrill et al., 2012).
Multiple studies conducted have shown that supplementation of D.
flagrans results in substantial (>80%) reduction of both infective larvae collected from feces as well as reduction of infective larvae on pasture (Waller et al., 1994;Larsen et al., 1998;Waller et al., 2001;Chandrawathani et al., 2004). Dose responses have been demonstrated, however, and continuous feeding of D. flagrans is needed throughout the grazing season in order for sustained effects (Larsen et al., 1998;Ketzis et al., 2006). This has been seen as a potential drawback to using D. flagrans as a control method, due to the impracticality of daily dosage for animals out on pasture. Suggestions have been made for administration methods, such as voluntary block feeding, which showed significant reductions of larval numbers in fecal cultures in sheep with even modest and irregular block consumption (Waller et al., 2001). A recent study in Australia to determine the effects of an investigational supplement known as Bioworma (providing 3 x 10 4 viable chlamydospores of D. flagrans strain IAH 1297/kg bodyweight (b.w.)/ day), showed a decreased number of parasitic nematode larvae on pasture and in turn, lower infections found in tracer-lambs grazing on the pasture (Healey et al., 2018). This study still did not address the impracticality of daily dosage, as the supplement had to be fed to the sheep daily for proper dosage. A bolus administration that would allow a slow, extended release of fungal spores to be released could be a potential option to improve the practicality of this control method .

Anthelmintic plants
Previous research has shown that condensed tannins, also known as proanthocyanidins, can have an anthelmintic effect on GIN . The discovery that plant secondary compounds, including proanthocyanidins (PAC), suppress GIN infections has provided promise for alternative methods of GIN control . A warm-season perennial legume, sericea lespedeza (Lespedeza cuneata), has been extensively researched and found to have anti-parasitic effects Terrill et al., 2012;. Previous feeding trials have shown that consumption of sericea lespedeza can reduce fecal egg counts by 67-98% , while also reducing the abomasal worm count of H. contortus  The cranberry contains an abundance of bioactive compounds including proanthocyanidins (PAC), anthocyanins, flavonols, and phenolic acid  and has been shown to have many health benefits in mammals including bacterial anti-adhesion , inhibition of tumor growth (Neto et al., 2006), decreased risk of cardiovascular disease (Porter et al., 2001), and inhibition of certain gastrointestinal diseases (Su et al., 2010;. The cranberry vines (stems and leaves) where the fruit grows contain high levels of bioactive compounds including PAC and bioactive compounds as well . When cranberry bogs come out of winter dormancy there is an annual pruning of vines to prepare for the next growing season, providing a readily available source of the vines which are not currently marketed . For these reasons, cranberry vines are an ideal test candidate as an alternative anthelmintic option for Northeast producers of small ruminants.

In vitro/In vivo screening results
The use of cranberry vines as an alternative anthelmintic has been investigated recently as an option for northeast producers. A study by  provided in vitro evidence of anthelmintic activity of CV-PAC organic extracts as well as CV aqueous extracts (CV-AqE) against Haemonchus contortus L1 and adult worm motility and some activity of CV-AqE against egg hatching . These results suggest that the PAC in the cranberry vines is not the only bioactive compound present and responsible alone for the anti-parasitic effects. A preliminary in vivo study was also carried out to determine the effects of oral administration of CV on lambs infected with H. contortus, showing a suppression of FEC in the first two weeks post-treatment with CV ( . Due to the promising in vitro and in vivo results from the Barone et al. study (2018), further testing to evaluate the in vivo effects of cranberry vine on GIN through feeding trials is warranted and form the basis for the current study.

Summary and conclusion
Previous studies suggest that both cranberry and cranberry vines can have health benefits due to their abundance of bioactive compounds. The  There was no significant effect of treatment (p=0.43) or treatment by time (p<0.09) on FEC, although the FEC of the two groups did start to diverge at week 5, and the trial ended prior to determining whether this divergence would reach significance. As would be expected, there was an effect of week on FEC (p < 0.01) as the trickle infection matured during the course of the trial.
There was no effect of treatment (p=0.79) or treatment by time (p=0.57) on packed cell volume. There was a difference by week (p<0.001) as the PCV decreased over time as the infections matured. Average daily gain over the 6- week trial of the two groups was not different (p = 0.52). There was a treatment*week effect (p = 0.0003); however, all significant changes were found within the control group due to a more rapid gain of weight and there was no significant difference between the treatments at any week. There was no difference in worm burden between control (3113 ± 554, n=7) and CV fed lambs (2687 ± 502, n=7) (p = 0.58). CV has shown anthelmintic potential but further studies are needed to improve palatability to increase consumption.

Introduction
Haemonchus contortus, commonly known as the barber pole worm, is the most pathogenic species of gastrointestinal nematode (GIN) infecting small ruminants, causing issues such as anemia, reduced nutrient absorption, production losses, and in severe cases, death. These GIN are a major economic concern for producers worldwide, causing a substantial impact on farm production and profitability . When compared to other GIN, H.
contortus is the most fecund, with female worms laying up to 10,000 eggs per day contributing to rapid pasture contamination . Larval and adult stages of H. contortus are found in the abomasum and abomasal mucosa where they consume up to 30 µL of blood per day, leading to anemia and in acute cases, death ). Due to the pathogenicity and economic impact associated with H. contortus, control is a major concern for sheep producers.
Over-and improper use of commercially produced chemical dewormers has led to the development of anthelmintic resistance in H. contortus as well as other GIN, creating the need for alternative control methods . In sheep and goats, prevalence of multiple drug resistance is remarkably high, and thus threatens the viability and sustainability of the industries Cezar et al. 2010;. The discovery that plant secondary compounds, including proanthocyanidins (PAC), suppress GIN infections has shown a possible method for alternative GIN control ).
The American cranberry (Vaccinium macrocarpon), native to New England, contains high levels of bioactive compounds, including proanthocyanidins .
Cranberry vines and leaves, also rich in PAC , are

Cranberry Vines
Clippings from bogs of 'Mullica Queen' cranberry owned by the A.D.
Makepeace Company (Wareham, MA, USA) were collected following pruning in spring 2017. Pruned vines were transported to the University of Rhode Island, then washed to remove dirt and debris before they were placed into burlap bags, and dried at 22 °C in cabinet dryers with continuous exhaust.
Moisture levels were monitored every other day by calculating weight changes to ensure that the vines were fully dehydrated when removed from the cabinet dryers. Vines were considered to be dry when the weight change was <1% from the previous weight recorded.

Cranberry vine pellet (CVP)
After drying, the CV was chopped into a powder using a hammer mill

Study Design
The study design for this feeding trial is depicted in Figure 1.  were sent to the abattoir and abomasal contents were collected for worm recovery, quantification, and identification of developmental stage and sex.

Analyses of Feed
Random grab samples of feed were collected daily to make weekly composite samples of CVP, grain, and hay. Weekly composite samples were used to create a composite sample for the full duration of the trial. Samples were analyzed by Dairy One Cooperative, Inc. (Ithaca, NY, USA) ( Table 1).  (Skantar et al., 2005;. All L3 were stored at 4°C for less than three months when used for infection.

Blood and fecal analysis
Blood samples were collected weekly via jugular venipuncture into sterile EDTA vacutainer tubes (BD, Franklin Lakes, NJ, USA) and PCV was determined by microhematocrit centrifugation at 35,720 x g for three minutes within 12 hours of sample collection. Rectal fecal samples were collected weekly for FEC determination according to the modified McMaster technique with a detection limit of 50 epg .

Analysis of parasite burden
At the abattoir, the abomasum of each lamb was opened along its greater curvature and a 10% aliquot of the contents were collected and fixed with equal volume of 10% formalin buffered saline. Total worm burdens were estimated by extrapolating the number of worms in the 10% aliquots. All counted worms were also sexed. The emptied abomasa were individually soaked overnight in biological saline at 37°C to allow immature larvae to migrate out of the abomasal folds. A 10% aliquot of wash contents were collected for quantification of larval burden.

Statistical analysis
Weekly FEC, PCV, and average daily gain were analyzed using a mixed procedure in SAS with repeated measures and means separated with Tukey's post-hoc test (SAS Inc., Cary, NC). Each multi-variable model included terms treatment, week, and two-way interactions. Worm and larval burden were analyzed using a student's t-test in SAS. Significance was defined as p ≤ 0.05. Results are reported as mean ± SEM.

Consumption of CVP
Consumption of CVP was determined in treatment lambs by monitoring orts for the duration of the trial. Lambs were offered 600g of CVP per day as a supplement added to their usual grain ration of 900g of 16% commercially produced pellet. During weeks 0 and 1, consumption of the pellet was extremely limited, averaging 68g of CVP consumed per lamb per day ( Figure   2). Palatability of the CVP varied greatly between lambs through the entire trial, with an average consumption of 281g (range 166 to 430) after six weeks of supplementation, approximately half of the 600g that was offered.

Effect of CVP on fecal egg counts
There was no effect of treatment (p=0.43) or treatment by time (p<0.09) although the FEC of the two groups did start to diverge at week 5, and the trial ended prior to determining whether this divergence would reach significance. As would be expected, there was an effect of week (p < 0.01) as the trickle infection matured during the course of the trial. contortus. Lambs were orally administered 5000 H. contortus L3 four weeks prior to the start of the feeding trial at week 0. Lambs were fed individually one of two diets: control (0g CV) or CVP (600g CV) for the duration of the trial.
Fecal egg counts (eggs per gram) were monitored weekly for the duration of the trial.

Effect of CVP on packed cell volume
There was no effect of treatment (control: 26 ± 0.4 %; CVP: 27 ± 0.4; p=0.79) or treatment by time (p=0.57) on PCV. There was a difference by week (p<0.001) as the PCV decreased over time. The PCV across treatments at weeks 0 and 2 was greater than week 5 (p ≤ 0.02); and at weeks 0 through 4 was greater than week 6 (p < 0.03). Average daily gain over the 6-week trial of control (0.13 ± 0.04 kg/day) and CVP (0.17 ± 0.08 kg/day) groups was not different (p = 0.52) when comparing CVP to control. There was a treatment*week effect (p = 0.0003); however, all significant changes were found within the control group due to a more rapid gain of weight ( Figure 4). There was no significant difference between the treatments at any week. contortus. Lambs were fed individually one of two diets: control (0g CV) or CVP (600g CV) for the duration of the trial.

Effect of CVP on worm burden and larval burden
The mean number of worms recovered from the control diet lambs was 2179 ± 55 worms, and the CVP lambs had a mean of 1881 ± 50 (p = 0.58) ( Figure 5). There was no difference between the percentage of females in the control and CVP groups (51 ± 2 vs 49 ± 2 respectively; p = 0.57). Effect of CVP on larval burden was not included in the results, as counts in soaks of control animals were insignificant, and consequently not completed.

Discussion
Supplementing CVP into the diet had an apparent suppressive effect on FEC by the end of the six-week trial, with no difference observed in PCV or total worm burden. Although palatability of the CVP greatly limited consumption, the results suggest that CV may have an anti-parasitic effect in vivo. The objective of this study was to expand upon in vitro screening results of cranberry vine extracts which showed anthelmintic activity against L1 and adult worm motility as well as some activity against egg hatching . The previous study also looked at the effect of orally administered cranberry vine powder, which appeared to produce slight suppression of FEC; however, limited quantities of the powder only allowed for a 3-day treatment therefore dose response or effects of feeding duration could not be investigated . Experimentally infected lambs were utilized for both the previous and current study. In contrast to the study by , lambs in the current study were subjected to a trickle infection after an initial bolus infection of H. contortus L3. The trickle infection allowed for simulation of reinfection that would occur in natural infections where animals were continuously exposed to low levels of infective larvae on pasture Conflicting results have similarly been observed when studying sericea lespedeza pellets. In one study an effect was observed after one week of feeding  and in another study differences were not observed until day 28 after treatment started ). The discrepancies have also been seen in studies using sericea lespedeza hay, where one study observed a difference in FEC after as little as one week , and another study did not show a difference until day 21 (Terrill et al. 2009). It has been suggested that these differences could be due to differences in GIN species present, season when treatment occurred, or an unknown mechanistic effect based on the plant secondary compounds present in the treatment . The process of sundrying, grinding, or pelleting also may have a negative impact on anthelmintic activity of bioactive forages by decreased extractability and reactivity of tannins in forages ; however, it has also been suggested that pelleting may actually increase effectiveness ). The current study, however, suggests that the pelleting process may have had a negative impact on the bioactivity of the CV, which could be due to excess heat incurred during the pelleting process. Although pelleting increases the ease of storage and feeding, further studies must be done, perhaps cooling the pellet during processing, to ensure that the process does not have a negative impact on the desired outcome for GIN control.
Packed cell volume was not affected by treatment in the current study, as both the treatment and control group PCV decreased over time due to the developing infections. These results are supported by findings for the total adult worm burden, which was not significantly different between the two groups by the end of the trial. The average PCV decreased from 28% to 24% from week 0 to 6 as the infections developed. Similar decrease in PCV over time has been observed in an H. contortus trickle infection, where PCV decreased from 30% to 20% as infections developed . The reduction in FEC that was observed toward the end of the current study could have been due to reduction of fecundity in the adult worms , although an effect on adult worms might have been observed if the CVP had been fed longer, which would likely affect both total worm burden and PCV as well ). Further investigation into the mechanism of action associated with feeding CV to parasitized animals must be done in order to maximize the desired benefit.
Average daily gain between treatment and control groups were not different (p=0.59) in the current study, although when comparing lamb weights across groups there was a treatment*week effect (p < 0.0003). There was no significant difference between the treatments at any week as the lambs were all growing and gaining weight. The treatment*week effect was likely due to the difference in gain patterns exhibited between the two groups; control lambs gained weight each week whereas treatment lambs fluctuated across weeks, eventually showing a net-gain. This fluctuation could be attributed to the low palatability of the CVP, which the lambs tried to eat around and sometimes also left a portion of their grain ration behind. It could also be due to the high ash-free neutral detergent fiber (aNDF) content in the CVP (64.5% of DM), as aNDF provides animals with bulk or fill and high values usually correspond to decreased dry matter intake (Santos et al. 2015). The 600g of CVP were given to lambs as a supplement in addition to their regular grain ration, and the lambs sorted their feed to consume minimal amounts of CVP.
Consumption by the end of the six weeks reached only about half of the 600g offered, therefore it is possible that the supplementation also affected consumption of the grain. Determining a method of CV supplementation that would not negatively affect weight gain in lambs is essential if the CV will be marketed for use by sheep producers.

Conclusions
This study provided limited in vivo evidence of anthelmintic efficacy using cranberry vine pellets as a feed additive for lambs infected with H.
contortus. Although there was no effect on packed cell volume or worm burden, the apparent suppression of FEC at weeks 5 and 6 in CV-fed lambs suggests that CV may have anti-parasitic potential but this would need to be confirmed by feeding the CV pellets for an extended period of time or increasing consumption levels of the pellet. Further studies are warranted to explore the use of CV as a feed supplement for the control of GIN in small ruminants.

CHAPTER 3
Anthelmintic weeks. The lambs were fed 1kg daily using a combination of CV pellets and commercially produced sheep pellets to achieve the targeted CV consumption.
Weekly weight, FEC, and packed cell volume (PCV) were determined. The FEC of lambs fed 500g CV were significantly lower than those of control lambs at week 1 (p = 0.02, 864 ± 205 vs 1807 ± 387; mean ± SEM) and week 2 (p = 0.03, 800 ± 273 vs 1671 ± 289). The FEC decreased in each group over the duration of the trial. There was no effect on PCV or weight gain. Further research is needed to establish the optimal time to feed CV for maximum anthelmintic effect against GIN.

Introduction
In the United States and globally, gastrointestinal nematode infection has been identified as the costliest disease to affect the sheep industry, representing great economic importance for producers Qamar et al., 2011). Gastrointestinal nematode (GIN) infections have been identified as one of the major limitations faced in pasture-raised production of small ruminants .
The costs associated with GIN are twofold, comprised of both direct expenses (decreases in production, decreases in gain, and host mortality) and indirect expenses (treatment costs and increased labor) (Torres-Acosta and Hoste, 2008).
Haemonchus contortus (the barber pole worm), Teladorsagia circumcincta (the brown stomach worm) and Trichostrongylus colubriformis (the bankrupt worm) are the most abundant gastrointestinal nematodes in sheep, and account for the greatest amount of economic and production losses (O'Connor et al., 2006). Haemonchus contortus is the most pathogenic species causing issues such as anemia, reduced nutrient absorption, production losses, and in severe cases death . When compared to other GIN, H. contortus is the most fecund, with female worms laying up to 10,000 eggs per day contributing to rapid pasture contamination . Larval and adult stages of H.
contortus are found in the abomasum and abomasal mucosa where they consume up to 30 µL of blood per day, leading to anemia and in acute cases, death ). Both T. circumcincta and T.
colubriformis can cause immune-mediated diarrhea in infected animals, and a mixed infection can lead to increased inflammatory cells in the small intestine (Williams et al., 2010). Decreased nutrient absorption and diarrhea associated with these GI worms lead to decreased gain and production losses as well.
The life cycle of T. circumcincta and T. colubriformis are similar to H.
contortus, but diarrhea or scouring in infected animals is an observable clinical symptom which is generally not associated with H. contortus, unless in mixed infections .
Historically, commercially produced chemical dewormers have been the go-to treatment for GIN infections because they were highly effective, safe to use and inexpensive (Šimpraga et al. 2015). Prophylactic treatment and overuse of these anthelmintics has led to the development of multiple drug resistance in H. contortus as well as other GIN, creating the need for alternative control methods Zvinorova et al., 2016). Previous The American cranberry (Vaccinium macrocarpon) and the cranberry plant contain high levels of bioactive compounds, including proanthocyanidins (Su et al. 2010;. Cranberry vines are pruned off of cranberry bogs when the plants come out of winter dormancy, generating a plentiful by-product of the industry . As such, the vines represent an ideal test candidate as a sustainable anthelmintic option for northeast sheep producers. This study was designed to expand upon in vitro screening results of cranberry vine extracts which showed anthelmintic activity against L1 and adult worm motility as well as some activity against egg hatching . The study by   weeks. Although palatability and consumption of the pellet was an issue, results showed that there appeared to be a suppression of FEC of lambs fed 600g of cranberry pellet for six weeks compared to control lambs (Chalut et al.,in preparation;chapter 2). This study aims to expand upon the previous in vivo trials by testing the anthelmintic efficacy of a nutritionally balanced 50% cranberry vine pellet on a mixed natural and experimental GIN infection in Dorset lambs.

Cranberry Vines
Clippings from bogs of 'Mullica Queen' and 'Howe' cranberry owned by the A.D. Makepeace Company (Wareham, MA, USA) were collected following pruning in spring and summer of 2018. Pruned vines were transported to the University of Rhode Island, then spread into windrows to dry at the University's Peckham Farm. The windrows were aerated and monitored daily until dry.

Cranberry vine pellet (CVP)
After drying, the CV was chopped into a powder using a hammer mill (Bison, MM20030000A, Aguascalientes, Mexico, 3/16" screen), and sent to Dairy One in Ithaca, NY for nutritional analysis. In order to ensure palatability a 50% CV pellet was formulated that was equivalent on a digestible dry matter basis to the commercially produced 16% sheep pellet used as the control pellet for this study (Pleasant View Farm, Somers, CT). The chopped CV was pelleted at Green Mountain Feeds (Bethel, VT). A sample of the CV pellet was sent to Dairy One in Ithaca, NY for nutritional analysis.

Study subjects
Feeding trials were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of the University of Rhode Island. Dorset lambs born and housed at the University of Rhode Island Peckham Farm, located in Kingston, RI were used for this study. Spring born lambs were weaned and raised indoors under trichostrongylid-free conditions until approximately five months of age when the trial began.

Study Design
At the start of the trial, twenty-one lambs (5 months old; weight 51 ± 3 kg (mean ± SEM; range 31 to 66)) were given daily access to a pasture lightly contaminated with GIN and remained on this pasture for the duration of the trial. After one week of being on pasture, an experimental infection of 5000 H.
contortus was superimposed on any natural infection in order to ensure an adequate infection for the feeding trial. Four weeks after the experimental infection, the lambs were stratified by fecal egg count (FEC) into three groups fed varying amounts of the cranberry vine pellet (CVP, 0, 250 or 500 g CVP/day). The groups were then balanced for sex and weight, with related animals (twins, triplets) split between groups. Control lambs were fed 0.9kg of a 16% crude protein commercially available pellet (Pleasant View Farms, Somers, CT, USA) and the treatment group rations fed were equivalent in digestible dry matter to the commercial pellet consumption. During the trial all lambs were individually fed twice daily one of three diets (n=7 each): control (0 CVP, 0.9 kg 16% commercial pellet/day), 250 g CVP/day (CVP 250), or 500 g CVP/day (CVP500) for 10 weeks. The lambs were sorted into their respective groups using a chute system to lead them into individual feeding stalls. When the lambs were finished eating, they were released back into their pen with continuous access to pasture. As stated above, the CVP, containing 50% CV, was formulated to be equivalent in digestible dry matter to the commercial 16% sheep pellet fed to the control animals. The animals in the CVP250 group received the balance of their ration in the 16% commercial sheep pellet.
The lambs also received 2.5 kg/lamb/day of grass hay, with water available ad libitum. Consumption of feed was monitored by measuring daily orts throughout the duration of the trial. Fecal egg count, packed cell volume (PCV), and weights were measured weekly. At the end of the ten-week trial lambs were sent to the abattoir and liver samples were collected from a subset of animals ( Figure 1).

Analyses of Feed
Random grab samples of feed were collected daily to make weekly composite samples of CVP, grain, and hay. Weekly composite samples were used to create a composite sample for the full duration of the trial. Samples were analyzed by Dairy One Cooperative, Inc. (Ithaca, NY, USA) ( Table 1).  (Skantar et al., 2005;. All L3 were stored at 4°C and were less than three months of age when used for infection.

Blood and fecal analysis
Blood samples were collected weekly via jugular venipuncture into sterile EDTA vacutainer tubes (BD, Franklin Lakes, NJ, USA) and packed cell volume (PCV) was determined by microhematocrit centrifugation at 35,720 x g for three minutes within 12 hours of sample collection. Fecal samples were collected weekly, directly from the rectum, for fecal egg count determination according to the modified McMaster technique with a detection limit of 50 epg .

Analysis of Liver Copper Levels
Due to increased levels of copper in the CV pellet, random liver samples were collected at the time of euthanasia from control and CV500 fed lambs and sent to Cornell University's Animal Health Diagnostic Center (Ithaca, NY) for toxicology analysis.

Statistical analysis
Weekly fecal egg counts, packed cell volume, and average daily gain were analyzed using a mixed procedure in SAS with repeated measures and means separated with Tukey's post-hoc test (SAS Inc., Cary, NC). Each multivariable model included terms treatment, week, and two-way interactions.
Liver copper values were analyzed using a student's t-test in SAS.
Significance was defined as p ≤ 0.05. Results are reported as mean ± SEM.

Consumption of CVP
Consumption of CVP was determined in treatment lambs daily by monitoring orts for the duration of the trial. Consumption of the CVP in both the CVP250 and CVP500 groups remained at or above 95% of the daily amount offered throughout the duration of the trial, therefore it was concluded that the 50% CVP was palatable to the lambs.

Effect of CVP on FEC
There was a treatment*week effect (p ≤ 0.001, Figure 2). There were differences within control (p ≤ 0.05) and CVP250 (p ≤ 0.01) lambs ( Figure   2). There were no differences over time within the CVP500 group and the treatments did not differ from each other at any week. varying amounts of cranberry vine pellet for 10 weeks (n=7 per group; Control 0 g/day, CVP250: 250 g CV/day and CVP500: 500 g CV/day).

Effect of CVP on packed cell volume
The mean PCV for all groups remained relatively stable over the course of the trial (Figure 3). The control group averaged 30 ± 1%, the CVP250 group averaged 29 ± 1% and the the CVP500 group averaged 29 ± 1 %over the entire course of the trial. There was a significant difference by week (p < 0.0001); however, there was no difference by treatment (p = 0.16), or treatment*week (p = 0.15). The PCV at weeks 0 and 1 was greater than the PCV at weeks 2 (p ≤ 0.01), 5 (p ≤ 0.03), and 6 (p ≤ 0.01) ( Figure 3).

Effect of CVP on weight and average daily gain
Average body weight gain over the 10-week trial of control (0.12 ± 0.04 kg/day), CV1 (0.11 ± 0.05 kg/day), and CV2 (0.10 ± 0.05 kg/day) groups were not significantly different throughout the duration of the trial (p = 0.24). There was no effect of treatment (p = 0.94) or treatment*week (p = 0.67) on weight over the course of the trial. There was a significant difference by week (p < 0.0001) as the weights increased over the course of the feeding trial while all of the lambs were growing and gaining weight ( Figure 4).

Effect of CVP on liver copper values
At the abattoir, liver samples were obtained from six randomly selected lambs -three from control and three from CV500-fed lambs to determine if their hepatic copper levels were affected by elevated copper concentration in the CV pellet. Liver samples were sent to Cornell University's Animal Health Diagnostic Center (Ithaca, NY) for toxicology analysis. Toxicology results showed that CV500-fed lambs had significantly higher copper levels than control-fed lambs (p ≤ 0.01).

Discussion
Under the conditions of this study, cranberry vine pellets were effective at suppressing a rise in FEC in lambs fed 500g of cranberry vine per day, as shown by the treatment*week effect (p ≤ 0.001). At the start of the trial, average FEC across all groups was 1343 ± 268 epg. Lambs had access to pasture for the duration of the trial, allowing for additional natural infection to develop. In both control and CV250 groups, FEC increased from week 0 to week 4 due to developing and additional infection. At week five, FEC in all three groups steadily declined throughout the duration of the trial with the exception of an increase in the CV250 group at week 7. The CV500 FEC values remained low from the start of the trial and were not significantly different throughout the trial, whereas the control and CV250 groups showed a clear rise in FEC before decreasing, suggesting that the CV500 diet had an early suppression effect on the infection, as early as one week following the initiation of feeding. This is similar to findings in a study that fed sericea lespedeza to naturally infected goats, with an experimental infection of H.
contortus superimposed, which showed a suppression of FEC starting one week following the start of feeding . This is also similar to results of a study that fed sericea lespedeza leaf meal pellets to goats, which also showed a decrease in FEC in the treatment group after one week of feeding with the suppression continuing through the entire four weeks of the trial period . These studies differ from the current study, however, as they both showed a significant decrease in FEC in the treatment group before suppressing FEC for the duration of feeding.
It is unclear what caused the suppression effect in the current study; however, in the pilot study by  The early FEC suppression observed in the current study and the study by  differ from the results of a prior in vivo study. In a previous study, lambs were experimentally infected with H. contortus and then given a trickle infection from weeks 0 to 3. The treatment group was offered 600g of a 100% CV pellet daily, although palatability greatly limited consumption to half that amount. The treatment and control group FEC behaved the same from weeks 0 to 4, and then began to diverge at weeks 5 and 6 where the control group continued to rise steadily and the treatment group started to decrease, suggesting the start of a suppressive effect (Chalut et al.,in preparation;chapter 2). These conflicting results have similarly been observed when studying sericea lespedeza pellets, where in one study they had an effect after one week of feeding  and in another study differences were not observed until day 28 after treatment started ). It has been suggested that these differences could be due to differences in GIN species present, season when treatment occurred, or an unknown mechanistic effect based on the plant secondary compounds present in the treatment . The pelleting process, and heat associated with it, may also have an effect on the bioactivity of the compounds found in these plants  can undergo a process known as hypobiosis where the L4 larvae go into a state of arrested development within the host for an extended period of time (Gatongi et al. 1998;O'Connor et al. 2006;). Hypobiosis occurs in H. contortus in the northern United States , therefore it is possible that some of the larvae consumed on pasture had entered a hypobiotic state toward the end of the trial.
Throughout the duration of the trial, packed cell volume remained within a high normal range in each group. Haemonchus contortus is a blood-feeding parasite which inhabits the abomasum, and one adult worm can consume up to 30 µL of blood each day, making anemia one of the primary concerns associated with heavy H. contortus infections . Anemia is a decline below the normal number of red blood cells, and has been defined as a packed cell volume ≤15-19%, depending on the exposure to H. contortus on pasture (Mederos et al. 2014;Kaplan et al. 2004).
The packed cell volume observed in each group would be expected to be within normal range due to the low to moderate levels of infection throughout the trial.
In the study by Chalut et al. (2017), palatability of a 100% cranberry vine pellet was a major limitation of the feeding trial, and lambs refused approximately half of the cranberry vine pellets that they were offered (Chalut et al.,in preparation). In order to address the issue of palatability associated with the bitterness of cranberry vine, likely due to the proanthocyanidins (Monagas et al. 2010), the current study formulated a 50% CVP to be both nutritionally equivalent to a commercially produced sheep pellet as well as palatable. The formulated CVP was well accepted by the lambs throughout the duration of the trial, showing that palatability was highly enhanced as evidenced by a lack of difference in the weight gain between treatment groups. The significant difference by week observed reflects the growth of the young lambs. The similar weight gain, as well as the nutritional analysis of the pellet, show that the 50% CVP formulated was in fact nutritionally equivalent to the commercially produced sheep feed, and could be fed to lambs as a primary diet.
Although the CVP was very close to the same as the commercially produced sheep feed, there was a slightly elevated copper level that was observed and not anticipated as the cranberry vine was analyzed prior to pelleting. Copper must be used cautiously in sheep because they are at high risk for copper toxicosis when levels build up in the liver (Terrill et al., 2012;. During week 8 of the study, one lamb from the group of CV500-fed lambs perished and a necropsy was performed to determine the cause of death. There was a concern that the lamb had potentially suffered from copper toxicosis due to the slightly elevated levels of copper in the CVP. The necropsy showed that the lamb had likely died as a result of an intestinal torsion; however, renal and hepatic toxicology was also performed at Cornell University's Animal Health Diagnostic Center (Ithaca, NY) for toxicology analysis. The toxicology report showed a normal renal copper concentration of 12.95ppm (reference range 12-25ppm dry matter) and an elevated hepatic copper concentration of 546.2ppm (reference range 120-500ppm dry matter). According to the toxicology report, although the liver copper value was higher than expected in this lamb, clinical copper toxicosis was unlikely to have occurred due to the low renal copper concentration.
In order to follow-up on these findings, three randomly selected lambs were chosen from both the control and CV2 groups to obtain post-mortem liver samples for toxicology analysis at the end of the trial. Toxicology results revealed that CV500-fed lambs had significantly higher copper concentrations (385 ± 49 ppm) than control lambs (128 ± 31 ppm) (p ≤ 0.01). The liver copper concentration was not high enough to cause copper toxicosis; however, the elevation observed suggests that re-formulation to account for the copper level would likely be necessary if the CVP were expected to be fed on a long-term basis. Due to the promising results observed in previous studies and this study, re-formulation and follow-up studies are warranted to better understand the effects of CVP on GIN.

Conclusions
This study provided further in vivo evidence of anthelmintic efficacy by using nutritionally balanced cranberry vine feed pellets for lambs naturally and experimentally infected with H. contortus and possibly other GIN species.
Lambs fed a high level of the cranberry pellet showed suppression of fecal egg counts throughout the beginning weeks of the trial when compared to groups fed low levels of cranberry pellet fed or no cranberry pellets (control). There was expectedly no effect on PCV in either group due to relatively low infections throughout the trial. The effect of CVP on total worm numbers was not evaluated because lambs in all groups appeared to lose their infections by the end of the study. The results, both observed in previous studies and this study, suggest that CV could be incorporated into the diet of sheep as a way to sustainably suppress GIN infections on farms in the northeast United States. Further studies are warranted to explore the dose-response associated with the use of CV, as well as the most appropriate timing for dosage to maximize effects.

CONCLUSIONS
The goal of this research was to determine the utility of a cranberry vine pellet in limiting gastrointestinal nematode infections in sheep. Previous research has shown in vitro and some limited in vivo evidence of anthelmintic activity of cranberry vine extracts and cranberry vine powder. The feeding trials used in this research were designed to follow-up and expand upon the previous results. In the previous study by , cranberry vine powder was orally administered to lambs using a drench gun to ensure consumption. The treatment was given for three consecutive days, and FEC in the treated lambs showed a slight suppression compared to control lambs.
The first feeding trial of the current study used cranberry vine powder to produce a 100% cranberry vine pellet to be fed as a supplement lambs experimentally infected with gastrointestinal nematodes. Throughout the entire trial, consumption of the pellet was extremely limited and only reached half of the 600g offered by the end of the six weeks. Even with limited consumption, the treatment group started to show a slight suppression of FEC at week 6 when compared to the control group. Due to the somewhat promising results observed, a second feeding trial was designed to address the issue of consumption, and palatability of the pellet was made a top priority.
In the second feeding trial of the current study, a 50% cranberry vine pellet was formulated to be both palatable and nutritionally balanced with the commercially produced 16% sheep pellet that was being fed to the lambs.
There were two treatment groups in this study, CVP250 that consumed 250g of cranberry vine per day and CVP500 that consumed 500g of cranberry vine per day. Consumption of the pellet was not an issue at any point throughout the trial, showing that the palatability issue from the first trial had successfully been addressed. This trial was also designed to study a natural GIN infection, versus the two previous studies that looked at experimental Haemonchus contortus infections; however, limited pasture contamination created the need for an experimental infection to be superimposed on the pasture infection. The FEC of the lambs fed 250 g of CVP and the control group initially rose from weeks 1 to 4 whereas there was no increase in FEC in the lambs fed 500 g of CVP over this same period. This would potentially be indicative of a reduction in fecundity or overall worm burden, which could be determined in future studies using larval cultures and post-mortem worm samples to calculate these parameters.
In order to better understand the effects that have been observed, as well as address issues that have arisen, further studies should include in depth investigation into the dose response of cranberry vine. In the first trial, a suppression effect appeared to occur when lambs were consuming around 300g of CV per day; however, in the second trial the FEC of lambs consuming 250g per day increased in a manner similar to the control group FEC, while the FEC of lambs consuming 500g per day did not change. Further studies should also include analysis of the effects of CV on larval and adult stages of GIN in vivo using scanning electron microscopy, in order to further our understanding of the mechanism of action of CV on a gastrointestinal nematode infection. Furthermore, future studies should look into the optimal feeding time for CV to determine whether the utility could be increased if fed prior to infection rather than after an infection is already established.
To date, cranberry vine has shown modest anthelmintic potential against small ruminant GIN both in vitro and in vivo. The effects of CV on GIN observed are not comparable to the use of commercially produced anthelmintics, but it is possible that it could be used in conjunction with other methods to control GIN infections as well as reduce pasture contamination and reinfection. The mechanism of action and the corresponding bioactive compounds that are responsible for the observed effects are still unknown and require further investigation.
9. Adults will be identified by sex and stage of development.

Cornell University Toxicology Reports
Toxicology lamb 1810 Post-mortem toxicology from control and CVP500 lambs